Photodynamic therapy (PDT) is a multicomponent cancer treatment in which tumors are exposed to lethal singlet oxygen ('O2)-mediated photooxidative stress induced by a localized sensitizing drug. Much has been learned about mechanism of tumor cell photokilling by various sensitizers, apoptosis (programmed cell death) occurring in some cases and necrosis (non-programmed death) in others. However, the role of metabolic and environmental factors in PDT-induced apoptotic vs. necrotic cell death are still not well understood. Studies supported by the existing grant have focused on the effects of nitric oxide (NO) in this regard. Using breast tumor cells metabolically sensitized with protoporphyrin IX (PpIX), we found that (i) NO delivered during irradiation (NO-now) protected against necrotic photokilling by inhibiting free radical (chain) peroxidation of plasma membrane (PM) lipids; importantly, residual killing was switched from necrosis to apoptosis; (ii)NO delivered much earlier and no longer present during irradiation (NO-then) inhibited photokilling as well, preliminary data suggesting involvement of an iron signalingmechanism. In a liposome system, NO also protected PpIX from photodegradation, thus prolonging its 'Degenerating lifetime. The proposed studies will delve more deeply into these novel effects of NO with the following hypotheses proposed: (a) NO-now and NO-then generated by neighboring microvascularcells can enhance tumor cell resistance to PDT killing; (b) By inhibiting PM lipid chain peroxidation, NO-now can foster apoptosis by reducing ion pump inactivation and membrane permeabilization, thereby supporting pro-apoptotic energy metabolism; (c) By also protecting membrane- bound sensitizer from free radical-mediated degradation, NO-now can result in a "selection" for pro-apoptotic !O2 targets. The proposed in vitro studies for testing these hypotheses will involve model membranes, two human breast tumor lines (COH-BR1, MCF-7), PpEX and merocyanine 540 (MC540) as sensitizers, chemical and cellular (macrophage, endothelial) NO donors, and techniques such as fluorescence microscopy, spectrofluorimetry, immunoblotting, electrophoretic mobility shift assays, and high-performanceliquid and thin layer chromatography with electrochemical and phosphorimaging detection, respectively. The specific plan is to investigate (i) sensitizer protection by NO-now with prolonged !O2 photogeneration in model systems and cells; (ii)ability of NO-now to facilitate apoptotic photokilling while inhibiting necrosis; (iii) mechanisms by which NO-now accommodatesapoptosis; (iv) characteristics of NO-then-induced photoresistance; and (v) underlying mechanisms of NO-then-induced resistance. Although significant NO is produced by macrophages and endothelial cells in tumor vascular systems, little isknown about how it might impact PDT efficacy. These studies will provide important new insights along these lines, and in the case of NO-now may suggest novel approaches for accommodating apoptosis in PDT,this end-point beingpreferred over necrosis because inflammation is minimized.